The goals of Project 3 are to define the mechanisms of herpes simplex virus (HSV) regulation of host innate immunity. Innate immune responses are activated by receptors recognizing microbial macromolecules, which elicit the expression of cytokines, chemokines, and type I interferons that have antiviral activity and/or stimulate the adaptive responses to the microbes. Microbes, in turn, have evolved mechanisms to evade or modulate the innate and adaptive immune responses. HSV infection can block interferon regulatory factor 3 (IRF-3) signaling (Melroe, DeLuca, and Knipe, 2004; Melroe et al., 2007) and Toll-like receptor 2 (TLR2) signaling through a nuclear viral protein, ICPO (Kurt-Jones et al., in preparation). Second, we have shown that the HSV Us3 protein can also block innate signalling. In this project we will test our hypothesis that HSV nfection down-regulates host innate responses, and that this contributes to pathogenesis in animal models and in human infections. Our specific aims are: 1. To define the mechanisms by which HSV ICPO blocks IRF-3 signaling and TLR-2 signaling in collaboration with Project 1 (Kurt-Jones) and Project 2 (Fitzgerald) by defining the mechanism(s) by which ICPO decreases the level of nuclear NF-KB, determining if HSV ICPO interacts directly with IRF-3 and/or CBP/p300 and sequesters these factors at nuclear sites away from host promoters, and mapping the portion(s) of ICPO needed for the interactions and effects on signaling. 2. To define the in vivo effects of ICPO regulation of innate immunity in murine systems in collaboration with Projects 1 (Kurt-Jones) and 4 (Finberg) by construction of mutant recombinant viruses that are defective for each of these two regulatory mechanisms, and testing the hypothesis that ICPO effects on innate immunity play a role in the virulence and ability of the virus to establish latent infection in a mouse model system. 3. To define the mechanism by which the HSV virion protein US3 downregulates innate signaling pathways. Preliminary data indicate that US3 causes the degradation of adaptor molecules in transfected cells and blocks signaling. In this aim we will determine the mechanism by which US3 blocks TLR signaling, determine the effect of US3 mutations on HSV infection and latent infection in mice, and define the effects of US3 on immune responses to HSV. 4. To determine the role of HSV evasion of innate immunity in human pathogenesis by: defining the effect of HSV genetic variability on innate immunity control through the genetic analysis of HSV clinical isolates that cause defined levels of disease in humans. In this aim we will clone the ICPO and US3 genes from HSV-2 and HSV-1 clinical isolates and determine their effect on different allelic forms of TLR2. These studies may define ways to improve herpes vaccines and vaccine vectors and new herpes therapeutic approaches. RELEVANCE (See instructions):